Sulphonamide inhibition studies of the β-carbonic anhydrase GsaCAβ present in the salmon platyhelminth parasite Gyrodactylus salaris

Abstract A β-class carbonic anhydrase (CA, EC 4.2.1.1) present in the genome of the Monogenean platyhelminth Gyrodactylus salaris, a fish parasite, GsaCAβ, has been investigated for its inhibitory effects with a panel of sulphonamides and sulfamates, some of which in clinical use. Several effective GsaCAβ inhibitors were identified, belonging to simple heterocyclic sulphonamides, the deacetylated precursors of acetazolamide and methazolamide (KIsof 81.9–139.7 nM). Many other simple benezene sulphonamides and clinically used agents, such as acetazolamide, methazolamide, ethoxzolamide, dorzolamide, benzolamide, sulthiame and hydrochlorothiazide showed inhibition constants <1 µM. The least effective GsaCAβ inhibitors were 4,6-disubstituted-1,3-benzene disulfonamides, with KIs in the range of 16.9–24.8 µM. Although no potent GsaCAβ-selective inhibitors were detected so far, this preliminary investigation may be helpful for better understanding the inhibition profile of this parasite enzyme and for the potential development of more effective and eventually parasite-selective inhibitors.


Introduction
We have recently reported the cloning and characterisation of a b-class carbonic anhydrase (CA, EC 4.2.1.1) encoded in the genome of Gyrodactylus salaris, GsaCAb 1 , a platyhelminth (flatworm) parasite attacking various fish species 2,3 . The Atlantic salmon (Salmo salar) is particularly sensitive to this parasite, which produced catastrophic losses in fish farms in Scandinavian countries and elsewhere, starting with the 1970s [3][4][5] . By releasing proteolytic enzymes, the parasite attaches on the fish gills, fins or skin inducing the formation of wounds, which favour the emergence of infections, with debilitation and eventual death of the infected animals 5,6 . There are no effective drugs for the treatment of this parasitic disease, although a variety of inorganic salts, synthetic compounds/drugs (e.g., praziquantel, levamisole, mebendazole and toltrazuril) and other approaches (manual removal of the worms) have been investigated, with rather unsuccessful results 7 . Furthermore, many of these compounds/drugs induce serious host toxicity, raising thus significant human health concerns if such fish is to be consumed 7 . Thus, as for other platyhelminth parasites producing infection in vertebrates including humans, such as Schistosoma haematobium 8 or Schistosoma mansoni 9-11 there is a stringent need of alternative drug targets and efficient compounds to treat these infections.
CAs are well known drug targets for the management of human diseases [12][13][14][15] , with their inhibitors acting as diuretics 16 , antiepileptics 17 , antiglaucoma 18 , antiobesity 19 and antitumor agents 20 . In the last decade, CAs from pathogens started to be considered as possible targets for the development of antiinfectives, for the management of diseases provoked by bacteria 21 , fungi 22 , protozoa 23 and worms 10,11,24 . In the previous work 1 we have shown that GsaCAb has a significant catalytic activity for the physiologic, CO 2 hydration reaction, with a k cat of 1.1 Â 10 5 s À1 and a k cat /K m of 7.58 Â 10 6 M À1 Â s À1 . Furthermore, inorganic anions, a well-known class of CA inhibitors (CAIs) 14,15 inhibit the enzyme in the millimolar range, as for other aand b-CAs investigated for their interaction with such modulators of activity 14 . Among the investigated such inhibitors, sulfamide (K I of 81 mM) and sulphamic acid (K I of 6.2 mM) showed the most efficient inhibitory action 1 . Both of them incorporate the SO 2 NH 2 moiety found in the most investigated class of CAIs, the aromatic/heterocyclic sulphonamides and their isosteres (sulfamates, sulfamides) 14,15 . Thus in this work we report GsaCAb inhibition studies with a panel of such compounds, many of which are clinically used drugs ( Figure 1).

Chemistry
Compounds 1-24 and AAZ-HCT were commercially available, highest purity reagents from Sigma-Aldrich (Milan, Italy) or were synthesised as previously reported 25 .

Production of b-CA recombinant protein
Protein production was carried out according to the previously reported protocol 1 .

Ca activity and inhibition measurements
An Applied Photophysics stopped-flow instrument has been used for assaying the CA catalysed CO 2 hydration activity 26   at a concentration of 0.2 mM was used as pH indicator, working at the absorbance maximum of 557 nm, with 10 mM TRIS (pH 8.3) as buffer, and in the presence of 10 mM NaClO 4 for maintaining constant the ionic strength, following the initial rates of the CA-catalysed CO 2 hydration reaction for a period of 10-100 s. The CO 2 concentrations ranged from 1.7 to 17 mM for the determination of the kinetic parameters and inhibition constants. For each inhibitor, at least six traces of the initial 5-10% of the reaction have been used for determining the initial velocity. The uncatalyzed rates were determined in the same manner and subtracted from the total observed rates. Stock solutions of inhibitors (10-20 mM) were prepared in distilled-deionized water and dilutions up to 0.01 mM were done thereafter with the assay buffer. Inhibitor and enzyme solutions were preincubated together for 15 min at room temperature prior to assay, in order to allow for the formation of the enzyme-inhibitor complex. The inhibition constants were obtained by non-linear least-squares methods using PRISM 3 and the Cheng-Prusoff equation, whereas the kinetic parameters for the uninhibited enzymes from Lineweaver-Burk plots, as reported earlier 27,28 , and represent the mean from at least three different determinations. GsaCAb concentration in the assay system was of 11.9 nM.

Results and discussion
GsaCAb shows catalytic properties for the physiologic reaction similar to those of the slow human isoform hCA I, being however slightly less effective as a catalyst compared to hCA I (Table 1). On the other hand, it should be stressed that many CAs are among the most effective catalysts known in nature 14,15 , and even this level of activity is in fact quite significant.
We have investigated the inhibition profile of GsaCAb with a panel of sulphonamides and sulfamates ( Figure 1) known to effectively inhibit many classes of CAs, with some of these derivatives being clinically used drugs for decades, in the treatment of a multitude of diseases, as shown in the introduction. The names of the relevant drugs are reported in Figure 1, and as mentioned above, they are used as diuretics, antiglaucoma drugs, antiepileptics or for the management of other disorders connected with CA activity disbalances 14,15 . The GsaCAb inhibition data with these compounds, as well as those for hCA I and II (for comparison reasons), are shown in Table 2.
As seen from Table 2, where the inhibition data of the human a-class isoforms hCA I and II were also included for comparison, all investigated sulphonamides/sulfamates inhibited GsaCAb, with inhibition constants raging between 81.9 nM and 24.8 mM. The following structure-activity relationship (SAR) should be noted regarding the inhibition data of Table 2: i. The most effective GsaCAb inhibitors were compounds 13 and 14, the deacetylated precursors of acetazolamide and methazolamide, which showed K I values of 81.9-139.7 nM, which is 5.1-5.6 times a better inhibitory activity compared to the clinically used derivatives AAZ and MZA ( Table 2). As seen in Table 2, these precursors are less effective as hCA I and II inhibitors compared to the acetylated derivatives used as drugs. ii. A rather large number of derivatives, such as 1-3, 7, 15-20, 2-24, AAZ, MZA, EZA, DZA, BZA, SLT and HCT, showed less effective inhibition, but anyhow with K I s <1000 nM. The SAR is rather difficult to rationalise in this case as these compounds belong to very heterogeneous classes of sulphonamides, both aromatic (benzene sulphonamides) and heterocyclic derivatives. However, it seems that rather simple and elongated scaffolds lead to effective inhibition whereas the inclusion of bulkier substituents (e.g. in 21 compared to 22-24, or BRZ compared to DZA) is detrimental for the inhibitory activity. iii. Compounds showing low micromolar inhibition of GsaCAb were 4-6, 8-10, 21, DCP, BRZ, TPM, ZNS, SLP; IND, VLX, CLX and SAC. These compounds had K I s in the range of 1.63-9.1 mM. As above, they belong to a large number of diverse chemotypes in order to draw a rationalisation of their SAR. Saccharin, also being a medium potency inhibitor, is Table 1. Kinetic parameters for the CO 2 hydration reaction catalysed by aand b-class CA enzymes: the human cytosolic isozymes hCA I and II (a-class) at 20 C and pH 7.5 in 10 mM HEPES buffer, and GsaCAb (measured at 20 C, pH 8.3 in 20 mM TRIS buffer and 10 mM NaClO 4 ) are shown. Inhibition data with the clinically used sulphonamide acetazolamide are also presented.

Isozyme
Activity level Low-moderate 1.1 Â 10 5 7.58 Â 10 6 460.5 a From ref. [12,15]; b From ref. [1]. Table 2. Inhibition of b-CA from G. salaris and human isoforms hCA I and hCA II with sulphonamides 1-24 and the clinically used drugs AAZ-HCT, by a stopped-flow assay 26 . among the most selective ones for inhibiting GsaCAb over the human isoforms (Table 2). iv. 4,6-disubstituted-1,3-benzene disulfonamides 11 and 12 were the least effective GsaCAb inhibitors, with K I s in the range of 16.9-24.8 mM (Table 2). v. The inhibition profile of GsaCAb and hCA I/II are very different, obviously due to the fact that they belong to diverse genetic CA families. Unfortunately, no GsaCAb-selective inhibitors (over the hCAs investigated here) were detected so far.

Conclusions
The Monogenean platyhelminth Gyrodactylus salaris, a fish parasite of salmon and other economically relevant aquaculture fish species, encodes for a b-class CA, GsaCAb, which has been investigated here for its inhibition profile with sulphonamides/ sulfamates, as a possible antiparasitic drug target. We identified several effective GsaCAb inhibitors, belonging to simple heterocyclic sulphonamide derivatives, the deacetylated precursors of acetazolamide and methazolamide, which showed K I values of 81.9 À 139.7 nM. Many other simple benezenesulfonamides and clinically used agents, such as acetazolamide, methazolamide, ethoxzolamide, dorzolamide, benzolamide, sulthiame and hydrochlorothiazide showed inhibition constants <1 mM. The least effective GsaCAb inhibitors were 4,6-disubstituted-1,3-benzene disulfonamides, with K I s in the range of 16.9 À 24.8 mM. Although no GsaCAb-selective inhibitors were detected so far, this preliminary investigation may be helpful for better understanding the SAR for inhibition of this parasite enzyme and for the potential development of more effective and eventually parasite-selective inhibitors.

Disclosure statement
CT Supuran is Editor-in-Chief of the Journal of Enzyme Inhibition and Medicinal Chemistry. He was not involved in the assessment, peer review, or decision-making process of this paper. The authors have no relevant affiliations of financial involvement with any organisation or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.